The first aim of this project is to develop an innovative methodology for measuring radionuclide uptake in single cells using a standard flow cytometer. The rationale for this aim is that flow cytometry in its current form can only interrogate cellular states by detecting fluorescence emissions from single cells, a process that excludes small-molecule compounds that are neither intrinsically fluorescent nor can be labeled with a fluorophore. The novel method we plan to develop is aimed at studying how single cells interact with any small molecule in the context of improving our understanding of fundamental cancer biology as well as developing new molecular agents for cancer diagnosis and treatment. Many small molecules can be labeled with beta-emitting radionuclides such as 11C, 18F, 32P, 35S, 64Cu, and 124I, which make the proposed approach almost universal with respect to the range of molecules that can be utilized. However, detecting radionuclides within a flow cytometer poses a major challenge. Due to the high throughput, each cell can only be measured for a few milliseconds, which is too short for a significant number of radioactive decays to occur. Thus, we plan to use photostimulable phosphors (PSPs) to physically record and store the number of radioactive decays that occur within each single cell over a prolonged exposure. Using microfluidics technology, we will encapsulate radioactive single cells and PSP microcrystals inside calcium-alginate droplets. This will ensure that PSP crystals are uniquely associated with a single cell. After complete decay of the radionuclide label, these droplets will be flowed through a flow cytometer to retrieve the energy stored inside the PSP microcrystals, which is directly proportional to the number of radioactive decays that occurred within each single cell. Hence, this approach will allow us to measure radionuclide uptake in up to 100,000 single cells. The second aim of this project is to develop a complementary approach for measuring the dynamic exchange of radiolabeled molecules across the membrane of single cells. Measuring the time-varying uptake of small molecules inside of single cells will allow us to quantitatively estimate influx and efflux rates and thus the amount and activity of various membrane transporters and enzymes within the cells. However, this requires that the same single cells be measured repeatedly over time in a statistically robust fashion. Thus, using microfluidic technology, we plan to develop a hydrodynamic cell-trapping array that is bonded to a transparent scintillator plate to enable facile and sensitive quantitation of the time-varying concentration of a radionuclide in approximately 500 single cells. Together, these two complementary research aims will open entirely new research avenues for studying normal and abnormal molecular processes in single cancer cells, with high throughput (aim 1) and high temporal resolution (aim 2).